U.S. patent number 6,232,072 [Application Number 09/419,447] was granted by the patent office on 2001-05-15 for biopolymer array inspection.
This patent grant is currently assigned to Agilent Technologies, Inc.. Invention is credited to William D. Fisher.
United States Patent |
6,232,072 |
Fisher |
May 15, 2001 |
Biopolymer array inspection
Abstract
A method of fabricating a biopolymer array, and an apparatus
which can execute the method. droplets of fluid carrying the
biopolymer or a biomonomer are deposited onto a front side of a
transparent substrate. Light is directed through the substrate from
the front side, back through a substrate back side and a first set
of deposited droplets on the first side to an image sensor. The
substrate may be mounted by means of a chuck with parallel
channels, and a scanning operation of a droplet dispensing head,
light source, and sensor conducted in unison in the direction of
the channels.
Inventors: |
Fisher; William D. (San Jose,
CA) |
Assignee: |
Agilent Technologies, Inc.
(Palo Alto, CA)
|
Family
ID: |
23662309 |
Appl.
No.: |
09/419,447 |
Filed: |
October 15, 1999 |
Current U.S.
Class: |
435/6.11;
436/518 |
Current CPC
Class: |
B01J
19/0046 (20130101); B01J 2219/00351 (20130101); B01J
2219/00378 (20130101); B01J 2219/00527 (20130101); B01J
2219/00529 (20130101); B01J 2219/00585 (20130101); B01J
2219/00596 (20130101); B01J 2219/00605 (20130101); B01J
2219/00608 (20130101); B01J 2219/00626 (20130101); B01J
2219/00659 (20130101); B01J 2219/00704 (20130101); B01J
2219/00707 (20130101); B01J 2219/00722 (20130101); C40B
40/06 (20130101) |
Current International
Class: |
B01J
19/00 (20060101); C12Q 001/68 (); G01N
033/543 () |
Field of
Search: |
;435/6 ;436/518
;356/364,369 ;422/82.05 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Website http://www.packardinst.com/pr/pr899-2.htm, Press Release:
"New Options Optimize Arraying Technology", Aug. 1, 1999, Packard
Instrument Company, date downloaded--Oct. 13, 1999. .
Website http:/www.packardinst.com/prod_serv/biochiparrayer.htm,
"BioChip Arrayer: Automated DNA Microarray Fabrication", Packard
Instrument Company, date downloaded--Oct. 13, 1999..
|
Primary Examiner: Brusca; John S.
Assistant Examiner: Kim; Young
Attorney, Agent or Firm: Stewart; Gordon
Claims
What is claimed is:
1. A method comprising:
(a) depositing droplets of fluid carrying a biopolymer or a
biomonomer onto a front side of a transparent substrate so as to
fabricate a biopolymer array; and
(b) directing light through the substrate from the front side then
backward through the substrate and a first set of deposited
droplets on the substrate front side, to an image sensor.
2. A method according to claim 1 wherein the biopolymer is a
polynucleotide.
3. A method according to claim 2 wherein the polynucleotide is a
DNA.
4. A method according to claim 1 wherein the light passes through
the substrate from the front side at a position other than the
first droplet set before being reflected to pass backward through
the substrate and first droplet set, and wherein the light is
reflected at a position spaced from a back side of the substrate so
as to reduce at the sensor any shadow from any non first set
droplets on the front side through which the light may initially
pass before being reflected.
5. A method according to claim 4 wherein the light passes through
the substrate from the front side at an angle to a normal of the
front side, and passes backward through the back side and first
droplet set at a complementary angle to the normal.
6. A method according to claim 1 wherein light passes through the
first droplet set when passing through the substrate from the front
side, before being reflected to again pass through the first
droplet set.
7. A method according to claim 6 wherein the light is reflected at
a position spaced from a back side of the substrate.
8. A method according to claim 1 additionally comprising:
repeating step (b) for additional sets of the deposited droplets by
scanning the directed and reflected light across the front
side.
9. A method according to claim 8 wherein both a light source of the
directed light and the image sensor are scanned in unison across
the front side.
10. A method according to claim 8 wherein the droplets are
deposited as droplet sets by a deposition head, and wherein
multiple droplet sets are deposited by scanning the deposition head
across the front side.
11. A method according to claim 10 wherein the light source, image
sensor and deposition head are physically interconnected and are
scanned in unison across the front side.
12. A method according to claim 8 wherein the light is reflected
from a mirror which faces at least an area on a back side of the
substrate corresponding to an area on the front side across which
droplet sets are deposited.
13. A method according to claim 10 wherein:
the substrate is held in a chuck having multiple upstanding ribs to
support a back side of the substrate;
light is reflected from a mirror comprising multiple mirror
segments extending along channels defined between the ribs; and
the deposition head is scanned across the front side of the
substrate by scanning along the channels in turn.
14. A method according to claim 13 wherein:
a light source of the directed light and a camera which includes
the sensor, are positioned so as to direct and receive the
reflected light at complementary angles to a normal of the front
side such that light from the source initially passes through the
front side of the substrate at a position other than the first
droplet set before being reflected to pass through the first
droplet set;
the direction of light from the source to the sensor being on a
plane oriented in the direction of the channels; and
the source and sensor are scanned in a direction along the channels
in turn.
15. A method according to claim 14 wherein the plane is parallel to
the direction of the channels.
16. A method according to claim 10 wherein the deposition head is
adjusted toward or away from the front side of the substrate
independently of the sensor.
17. A method according to claim 10 additionally comprising, after
depositing droplets of at least the first droplet set,
re-positioning the substrate with respect to the light source and
image sensor such that the reflected light is received at the image
sensor after it has passed back through the first droplet set.
18. A method according to claim 1 additionally comprising exposing
the array to a sample to be tested, and interrogating the array
following exposure.
19. A method according to claim 1 additionally comprising exposing
the array to a sample to be tested at a first location,
interrogating the array, and communicating the result or a
conclusion based on the result to a location remote from the first
location.
20. A method according to claim 19 wherein the sample was obtained
from a remote location.
21. An apparatus for fabricating a biopolymer array,
comprising:
(a) a mount on which a transparent substrate can be mounted;
(b) a deposition head to deposit droplets of a fluid carrying the
biopolymer or a biomonomer onto a front side of the mounted
substrate;
(c) a light source to direct light through the mounted substrate
from the front side;
(d) a reflector to reflect light from the source which has passed
through the mounted substrate backward through the mounted
substrate and a first set of deposited droplets on the front side;
and
(e) an image sensor facing a front side of the mounted substrate to
receive the reflected light after it has passed back through the
first droplet set.
22. An apparatus according to claim 21 wherein the reflector
comprises a mirror facing a back side of the mounted substrate.
23. An apparatus according to claim 22 wherein the deposition head
comprises multiple pulse jets each of which can be electrically
activated to deposit a droplet.
24. An apparatus according to claim 23 wherein the volume of a
droplet delivered by a pulse jet on a single activation is between
0.1 to 1000 pL.
25. An apparatus according to claim 21 wherein the light source and
the image sensor, are positioned such that light from the source
initially passes through the front side of the substrate at a
position other than the first droplet set before being reflected to
pass backward through the substrate and first droplet set.
26. An apparatus according to claim 25 wherein the mirror is spaced
from the back side of the mounted substrate so as to reduce at the
sensor any shadow from any droplets on the front side through which
the light may initially pass before being reflected from the
mirror.
27. An apparatus according to claim 25 wherein the light source is
positioned to direct light through the substrate at an angle to a
normal of the front side, and the image sensor is positioned to
receive the light which has passed back through the first droplet
set at a complementary angle to the normal.
28. An apparatus according to claim 21 wherein the light source and
image sensor are positioned such that light from the source
initially passes through the front side of the mounted substrate at
the position of the first droplet set before being reflected to
again pass through the first droplet set.
29. An apparatus according to claim 28 wherein the mirror is spaced
from the back side of the substrate.
30. An apparatus according to claim 22 additionally comprising:
a transport system to scan at least one of the deposition head and
substrate across the other.
31. An apparatus according to claim 30 additionally comprising a
processor to control the transport system and cause the deposition
head to dispense multiple droplet sets in co-ordination with
relative movement of the deposition head and substrate.
32. An apparatus according to claim 22 additionally comprising a
transport system to scan both the light source and image sensor in
unison across the front side so that the reflected light can be
received at the sensor from additional sets of the deposited
droplets.
33. An apparatus according to claim 32 wherein the light source,
image sensor and deposition head are physically interconnected, and
the transport system scans the light source, image sensor and
deposition head in unison across the front side.
34. An apparatus according to claim 30 wherein:
the substrate mount comprises a chuck having multiple upstanding
ribs to support the back side of the substrate;
the mirror comprises multiple mirror segments extending along
channels defined between the ribs; and
the transport system scans the deposition head across the front
side of the substrate by scanning along the channels in turn.
35. An apparatus according to claim 34 wherein:
the light source and a camera which includes the image sensor, are
positioned so as to direct and receive the reflected light at
complementary angles to a normal of the front side such that light
from the source initially passes through the front side of the
substrate at a position other than the first droplet set before
being reflected to pass through the first droplet set;
the direction of light from the source to the sensor being on a
plane oriented in the direction of the channels; and
the source and sensor are scanned in a direction along the channels
in turn.
36. An apparatus according to claim 35 wherein the plane is
parallel to the direction of the channels.
37. An apparatus according to claim 22, wherein the light source,
image sensor and deposition head are physically interconnected, the
apparatus additionally comprising a transport system to scan the
light source, image sensor and deposition head in unison across the
front side.
38. An apparatus according to claim 37 wherein the transport system
adjusts the deposition head toward or away from the front side of
the mounted substrate independently of the sensor.
Description
FIELD OF THE INVENTION
This invention relates to arrays, particularly polynucleotide
arrays such as DNA arrays, which are useful in diagnostic,
screening, gene expression analysis, and other applications.
BACKGROUND OF THE INVENTION
Polynucleotide arrays (such as DNA or RNA arrays), are known and
are used, for example, as diagnostic or screening tools. Such
arrays include features (sometimes referenced as spots or regions)
of usually different sequence polynucleotides arranged in a
predetermined configuration on a substrate. The arrays, when
exposed to a sample, will exhibit a binding pattern. The array can
be interrogated by observing this binding pattern by, for example,
by labeling all polynucleotide targets (for example, DNA) in the
sample with a suitable label (such as a fluorescent compound), and
accurately observing the fluorescent signal on the array. Assuming
that the different sequence polynucleotides were correctly
deposited in accordance with the predetermined configuration, then
the observed binding pattern will be indicative of the presence
and/or concentration of one or more polynucleotide components of
the sample. Peptide arrays can be used in a similar manner.
Biopolymer arrays can be fabricated using either in situ synthesis
methods or deposition of the previously obtained biopolymers. The
in situ synthesis methods include those described in U.S. Pat. No.
5,449,754 for synthesizing peptide arrays, as well as WO 98/41531
and the references cited therein for synthesizing polynucleotides
(specifically, DNA). Such in situ synthesis methods can be
basically regarded as iterating the sequence of depositing droplets
of: (a) a protected monomer onto predetermined locations on a
substrate to link with either a suitably activated substrate
surface (or with a previously deposited deprotected monomer); (b)
deprotecting the deposited monomer so that it can now react with a
subsequently deposited protected monomer; and (c) depositing
another protected monomer for linking. Different monomers may be
deposited at different regions on the substrate during any one
iteration so that the different regions of the completed array will
have different desired biopolymer sequences. One or more
intermediate further steps may be required in each iteration, such
as oxidation and washing steps.
The deposition methods basically involve depositing biopolymers at
predetermined locations on a substrate which are suitably activated
such that the biopolymers can link thereto. Biopolymers of
different sequence may be deposited at different regions of the
substrate to yield the completed array. Washing or other additional
steps may also be used. Typical procedures known in the art for
deposition of polynucleotides, particularly DNA such as whole
oligomers or cDNA, are to load a small volume of DNA in solution in
one or more drop dispensers such as the tip of a pin or in an open
capillary and, touch the pin or capillary to the surface of the
substrate. Such a procedure is described in U.S. Pat. No.
5,807,522. When the fluid touches the surface, some of the fluid is
transferred. The pin or capillary must be washed prior to picking
up the next type of DNA for spotting onto the array. This process
is repeated for many different sequences and, eventually, the
desired array is formed. Alternatively, the DNA can be loaded into
a drop dispenser in the form of an inkjet head and fired onto the
substrate. Such a technique has been described, for example, in PCT
publications WO 95/25116 and WO 98/41531, and elsewhere. This
method has the advantage of non-contact deposition.
In either method of fabrication, glass or other transparent
material, is often used for the substrate. Such materials
particularly lend themselves to linking of a nucleotide of a
monomer or polymer. Further, in array fabrication, the quantities
of DNA available for the array are usually very small and
expensive. Sample quantities available for testing are usually also
very small and it is therefore desirable to simultaneously test the
same sample against a large number of different probes on an array.
These conditions require use of arrays with large numbers of very
small, closely spaced features. It is important in such arrays that
features actually be present, that they are put down accurately in
the desired pattern, are of the correct size, and that the DNA is
uniformly coated within the feature. Normally, in an automated
apparatus the features are deposited according to a target array
pattern.
However, every component in an array deposition apparatus are
subject to errors such as component failure or variances in its
operating parameters within, or sometimes even outside of, normal
tolerances for such component. For example, a dispensing head used
to dispense fluid droplets to form the array, may have one or more
jets which fail or which vary slightly in the size of the droplets
dispensed, the orientation of the jets with respect to one another,
or the orientation of the head itself in the apparatus may be
slightly off from a nominal position. Whatever the error source,
the result is that a target array pattern is not produced. That is,
there is a discrepancy between the target array pattern and the
actual array pattern deposited. These discrepancies can occur in
each cycle of the in situ process, or during deposition of
presynthesized polynucleotides.
The validity of the results of any test using an array, is
dependant on knowing where the features are on the carrier
substrate and if they were actually there on the substrate to begin
with. A line scan camera can be used to observe droplets after
their deposition during fabrication to reduce the possibility that
during array use in a test, a reaction did not occur because a
feature was missing or subject to some other error, thereby
resulting in a false test result. However, observing the droplets
during array fabrication can, as a practical matter, be difficult.
For example, it is difficult to obtain sufficient reflected light
from either the droplets or substrate surface to the camera sensor
as the droplets move past the line scan camera. The amount of light
the camera sensor is exposed to is inversely proportional to the
speed of the objects being viewed. The faster the objects move, the
less light reaches the camera, which can result in poor image
contrast for reliable feature imaging. While the fabrication speed
could be slowed to capture more light, this is undesirable from a
manufacturing perspective and so the line scan camera should
capture images at the running speed of the system. An additional
issue with obtaining sufficient light to the camera is that when
the substrate is glass or is otherwise transparent, and given that
the droplets themselves may be transparent and colorless, most of
the light will pass through the substrate and not make it back to
the camera.
It would be useful then, to provide a means by which arrays can be
fabricated by depositing droplets of monomer, polymer, or any other
material used during array formation, and in which the deposition
of the droplets can be accurately and rapidly observed even against
a transparent substrate. It would also be useful if any such means
is relatively simple to construct and offers little interference
with other components of a deposition apparatus.
SUMMARY OF THE INVENTION
The present invention realizes that relatively good images can be
obtained of deposited droplets of monomer, polymer (such as DNA,
RNA, or peptides), or other fluids deposited on one side of a
transparent substrate during array fabrication, by back lighting
those droplets (that is, by providing illumination from the back
side of the substrate). However, the present invention further
realizes that back lighting during array fabrication is not
particularly practical. For example, the substrate should
preferably be firmly and precisely held in a known position at all
times so that droplets will be deposited at least close to the
expected locations, while still being supported on a back side.
This implies some type of clamping and support structure across a
back side of the substrate thereby making the provision of any
lighting system across the back side during array manufacture,
difficult. Further, illuminating an entire back side of a substrate
could lead to undue heating which could adversely affect sensitive
biopolymers, given the intensity of illumination required. The
present invention realizes that back lighting a transparent
substrate during array fabrication can be accomplished by providing
a mirror on the back side and illuminating and imaging from the
front side on which droplets are deposited. The present invention
further realizes that scanning illumination on the front side can
avoid undue heating.
The present invention then, provides a method of fabricating a
biopolymer array (for example, a polynucleotide or, more
specifically, a DNA or RNA array). The method includes depositing
droplets of fluid carrying the biopolymer or a biomonomer onto a
front side of a transparent substrate. Light is directed through
the substrate from the front side, back through a substrate back
side and a first set of deposited droplets on the first side to an
image sensor. In this manner, the first set is "imaged". The light
may optionally pass through the substrate from the front side at a
position other than the first droplet set before being reflected to
pass back through the back side of the substrate and first droplet
set. Particularly, the light may pass through the substrate from
the first side at an angle to a normal of the first side, and pass
back through the back side and first droplet set at a complementary
angle to the normal. Alternatively, the light may pass through the
first droplet set when passing through the substrate from the first
side, before being reflected to again pass through the first
droplet set. In either event, the light is optionally reflected at
a position spaced from the back side.
The directing of light in the foregoing manner may be repeated for
additional sets of the deposited droplets by scanning the directed
and reflected light across the first side. This can, for example,
be accomplished by scanning both a light source of the directed
light and the image sensor in unison across the first surface.
Furthermore, the droplets may be deposited as droplet sets by a
head, and multiple droplet sets may be deposited by scanning the
head across the first side. Any deposited set may or may not be the
same set that is later imaged by the sensor as a set. Further, the
light source, image sensor and head are preferably physically
interconnected and are scanned in unison across the first surface.
The mirror preferably faces at least that area on the second side
corresponding to that area on the first side across which droplet
sets are deposited.
The substrate may, in one arrangement, be held in a chuck having
multiple upstanding ribs to support the second side of the
substrate. In this case, the mirror from which light is reflected
may include multiple mirror segments extending along channels
defined between the ribs. The head may be scanned across the first
side of the substrate by scanning along the channels in turn. The
direction of light from the source to the sensor may be on a plane
oriented along (and preferably parallel to) the direction of the
channels, and the source and sensor also scanned in a direction
along (and preferably parallel to) the channels. This is
particularly useful in the case where the light source and camera
are positioned at so as to direct and receive the reflected light
at complementary angles in the manner as described above. The head
may be adjusted toward or away from the first side of the substrate
independently of the sensor.
An apparatus which can execute a method of the present invention,
is also provided. Such an apparatus includes a mount on which a
transparent substrate can be mounted. A head is provided to deposit
the fluid droplets onto the front side of the mounted substrate. A
light source, reflector, and image sensor are also provided to
execute the steps required by them, as described above. The
apparatus may include other features as already described in
connection with the method. For example, the apparatus may further
include a transport system for the head, light source and image
sensor, so as to move them in a manner as described, preferably
including scanning in unison (with the head, light source, and
image sensor being preferably physically interconnected as
described above). A processor may also be provided to control the
transport system as required and cause the head to dispense
multiple droplet sets in co-ordination with relative movement of
the head and substrate. Other elements may include the substrate
mount in the form of a chuck, as described above.
Apparatus and methods of the present invention, can be also be used
to deposit droplets of any other fluid moiety or moieties, and
embodiments of the apparatus can be described by replacing
"biopolymer", or similar terms with "moiety". Also, methods of the
present invention can be executed without the deposition step in
the event that the droplets were previously provided on the
substrate.
The present invention can provide any one or more of a number of
advantages. For example, the deposition of the droplets can be
accurately observed against a transparent substrate. The imaging
arrangement is relatively simple to construct and offers little
interference with other components of a deposition apparatus, such
as a chuck of the type described. The light source and camera
scanning arrangement can offer rapid scanning with little
interference from chuck components, such as the described ribs.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of a substrate bearing multiple
arrays, as may be produced by a method and apparatus of the present
invention;
FIG. 2 is an enlarged view of a portion of FIG. 1 showing some of
the identifiable individual regions (or "features") of a single
array of FIG. 1;
FIG. 3 is an enlarged cross-section of a portion of FIG. 2;
FIG. 4 is perspective view of an apparatus of the present invention
without a mounted substrate;
FIG. 5 is a vertical cross-section of the apparatus of FIG. 4 with
a mounted substrate;
FIG. 6 is an end view of a chuck of the apparatus of FIG. 4;
and
FIG. 7 schematically illustrates an alternative arrangement for the
light source and sensor of the apparatus of FIG. 4.
To facilitate understanding, identical reference numerals have been
used, where practical, to designate identical elements that are
common to the figures.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
In the present application, unless a contrary intention appears,
the following terms refer to the indicated characteristics. A
"biopolymer" is a polymer of one or more types of repeating units.
Biopolymers are typically found in biological systems and
particularly include peptides or polynucleotides, as well as such
compounds composed of or containing amino acid analogs or non-amino
acid groups, or nucleotide analogs or non-nucleotide groups. This
includes polynucleotides in which the conventional backbone has
been replaced with a non-naturally occurring or synthetic backbone,
and nucleic acids (or synthetic or naturally occurring analogs) in
which one or more of the conventional bases has been replaced with
a group (natural or synthetic) capable of participating in
Watson-Crick type hydrogen bonding interactions. Polynucleotides
include single or multiple stranded configurations, where one or
more of the strands may or may not be completely aligned with
another. A "nucleotide" refers to a sub-unit of a nucleic acid and
has a phosphate group, a 5 carbon sugar and a nitrogen containing
base, as well as analogs (whether synthetic or naturally occurring)
of such subunits which in the polymer form (as a polynucleotide)
can hybridize with naturally occurring polynucleotides in a
sequence specific manner analogous to that of two naturally
occurring polynucleotides. For example, a "biopolymer" includes DNA
(including cDNA), RNA, oligonucleotides, and PNA and other
polynucleotides as described in U.S. Pat. No. 5,948,902 and
references cited therein (all of which are incorporated herein by
reference), regardless of the source. An "oligonucleotide"
generally refers to a nucleotide multimer of about 10 to 100
nucleotides in length, while a "polynucleotide" includes a
nucleotide multimer having any number of nucleotides. A
"biomonomer" references a single unit, which can be linked with the
same or other biomonomers to form a biopolymer (for example, a
single amino acid or nucleotide with two linking groups one or both
of which may have removable protecting groups). A biomonomer fluid
or biopolymer fluid reference a liquid containing either a
biomonomer or biopolymer, respectively (typically in solution). An
"addressable array" includes, for example, any one or two
dimensional arrangement of discrete regions (or "features") bearing
particular biopolymer moieties (for example, different
polynucleotide sequences) associated with that feature and
positioned at a particular location on the substrate (an
"address"). These features may or may not be separated by
intervening spaces. By "viewing" or "imaging" a set of droplets and
"sensors" for accomplishing such imaging, references using any
optical device to provide data on the whether the droplets are
present, and/or the location and/or size of the droplets. "Light"
includes visible or non-visible light (including infra-red or
ultraviolet). It will also be appreciated that throughout the
present application, words such as "upper", "front", "back", and
the like are used with reference to a particular orientation of the
apparatus with respect to gravity, but it will be understood that
other operating orientations of the apparatus or any of its
components, with respect to gravity, are possible. To "position"
one item with respect to another, includes moving either one or
both of them to obtain the desired relative positioning. "Fluid" is
used herein to reference a liquid. Reference to a "droplet" being
dispensed herein (whether from a pulse jet or otherwise), merely
refers to a discrete small quantity of fluid (usually less than
about 1000 pL) being dispensed at a given time (for example, upon a
single pulse of the pulse jet corresponding to a single activation
of an ejector) and does not require any particular shape of this
discrete quantity. Droplets may be dry, but more typically will
still be wet at the time they are inspected. However, it will be
understood that a given feature may be formed from one or multiple
pulses from one or multiple nozzles. A "set" may contain one or
multiple members. The head or substrate moving as droplets are
dispensed, includes actual movement during and/or between the
dispensing of multiple droplets. By one item being "remote" from
another is referenced that they are at least in different
buildings, and may be at least one, at least ten, or at least one
hundred miles apart.
Referring first to FIGS. 1-3, typically the present invention will
produce multiple identical arrays 12 (only some of which are shown
in FIG. 1), separated by inter-array regions 13, across the
complete front surface 11a of a single transparent substrate 10.
However, the arrays 12 produced on a given substrate need not be
identical and some or all could be different. Each array 12 will
contain multiple spots or features 16 separated by inter-feature
regions 15. A typical array 12 may contain from 100 to 100,000
features. All of the features 16 may be different, or some or all
could be the same. Each feature carries a predetermined moiety
(such as a particular polynucleotide sequence), or a predetermined
mixture of moieties (such as a mixture of particular
polynucleotides). This is illustrated schematically in FIG. 3 where
different regions 16 are shown as carrying different polynucleotide
sequences. Arrays of FIGS. 1-3 can be manufactured by in situ or
deposition methods as discussed above.
Referring to FIG. 4 the apparatus shown includes a mount for
substrate 10 in the form of chuck 20. A substrate 10 is mounted
onto chuck 20 in the manner illustrated in FIG. 5. Chuck 20 is a
vacuum chuck of generally rectangular configuration, and includes a
bottom plate 22, and a plurality of upstanding parallel linear ribs
24 which define a series of parallel rectangular channels 26
between them. Ribs 24 have upper surfaces 25 with openings which
communicate with a suitable vacuum source (not shown), such that
ribs 24 can hold against, and support, a second side 11b of a
mounted substrate 10 in the manner illustrated in FIG. 5. A mirror
27 is provided, in the form of multiple mirror segments 28
extending along the bottom of respective channels 26 between ribs
24, so as to be spaced from a back side 11b of a mounted substrate
10. Mirror segments may be defined by a metallized reflecting layer
on the back surface of a glass or other transparent substrate.
(although front surfaced mirrors could be used if desired). In this
situation it will be understood that reference to a "mirror" refers
to the actual reflecting layer.
The apparatus further includes two tracks 84 along which a first
frame member 80 can be precisely moved by means of a motor 86 (also
mounted on frame member 80) working against tracks 84 through a
track drive. Tracks 84, frame member 80, motor 86 and track drive,
act as components of a first transporter. Heads 40 are provided to
deposit droplets of biopolymer or biomonomer solution onto the
front side 11a of a mounted substrate 10. Heads 40 are mounted to a
third frame member 92 by a head retainer 44, third frame member 92
being slidable toward and away from chuck 20 on a second frame
member 88 fixedly mounted to first frame member 80. A suitable
motor and drive mechanism, which act as components of a second
transporter, are provided inside second frame member 88 to cause
such movement. Chuck 20 is also provided with a suitable motor and
drive mechanism (not shown), which act as components of a third
transporter, which can move chuck 20 in a direction 110. Thus, the
first and second transporters act as a transport system which can
scan heads 40 across front surface 11a of a mounted substrate 10 to
deposit multiple droplets of biopolymer of biomonomer solution.
Such scanning would normally be done in a row by row format. In the
row by row format heads 40 are first moved by the transport system
in the direction of axis 106, which movement is coordinated by a
suitably programmed processor (not shown) with firing of the pulse
jets of heads 40 to deposit a row of droplets in accordance with a
target array pattern. Note that by virtue of head 40 construction
as described below, a "row" will typically include multiple lines
of droplets. Substrate 40 is then moved by the transport system
parallel to axis 110, specifically in the direction of arrow 110a
at least the width of one row, and the process repeated. Of course,
other deposition formats could be used.
A light source 50 is mounted to first frame member 80 through block
100 to direct light through substrate 10 front the front side 11a.
Light source 50 includes a lens 54 and an optical fiber bundle 52
which communicates light in the visible region (substantially 400
nm to 700 nm) from a suitable source (not shown). A linescan camera
60 includes an adjustable focus lens 62 and a linear CCD or other
linear sensor 64 (see FIG. 5) oriented parallel to axis 110. Camera
60 (and hence sensor 64) are mounted to first frame member 80 by
being mounted on a side of block 100 opposite that of light source
50. Note that the block 100 is so constructed such that the angle a
between the light directed by light source 50 through mounted
substrate 10 (shown by axis 130 in FIG. 5) and a normal 104 to
first side 11a of mounted substrate 10, and the angle .beta.
between the light which is reflected light back through the back
side 11b (shown by axis 140 in FIG. 5) and normal 104, are
complementary angles (that is, of the same magnitude but in
opposite directions about normal 104). The total angle
(.alpha.+.beta.) should be kept as small as possible (such as less
than 50.degree. or even less than 40.degree.), limited only by the
physical size of the components, to provide a compact arrangement.
Furthermore, keeping the angle small limits the distance at which
mirror 27 needs to extend beyond the array being formed. Note also
that the direction of the light from light source 50 (as
illustrated by axis 130) and to sensor 64, lie on a common plane
which is oriented in the direction of channels 26 (specifically, by
being parallel to those channels 26).
The above apparatus is used to fabricate an array in the following
manner. It will be understood that all of the operations
particularly following mounting of the substrate 10, can be
controlled by a suitably programmed processor, such as a programmed
general purpose processor or any hardware/software equivalent. It
will be assumed that the heads have already been loaded with one or
more biopolymer or biomonomer solutions, that lens 62 is already
adjusted so as to be focussed on the front surface 11a of a mounted
substrate, and that the light source 50 and camera 60 are ON.
First, heads 40 are moved away from chuck 24 by the second
transporter causing third member 92 to slide upward (as viewed in
FIG. 4) on second member 88. Note that the above described
construction allows heads 40 to be adjusted toward or away from
chuck 20 (and hence first side 11a of substrate 10) independently
of movement of sensor 64. That is, camera 60 will not move during
such height adjustment of heads 40, and thus the focus of camera 60
is not affected by such height adjustment. Substrate 10 may then be
mounted on chuck 20 by placing it with its back side 11b in contact
with upper surfaces 25 of ribs 24. A vacuum is applied through ribs
24 to the openings so as to firmly retain second side 11b in
position supported against ribs 24. The substrate mounting
operation is then complete. Heads 40 may then have their height
readjusted to a suitable distance from first side 11a for
dispensing fluid droplets, by again using the second
transporter.
The processor then causes transport system to scan heads 40 across
the front side 11a of substrate 40 in unison with light source 50
and camera 60, in a row by row format as described above.
Simultaneously, the processor activates the pulse jets in heads 40
in a sequence to dispense multiple droplets in co-ordination with
relative movement of the head and substrate, in accordance with the
target array pattern. Light source 50 and sensor 64 are positioned
such that sensor 64 images a set of droplets (referenced as a
"first set"), specifically a line of droplets, forming part of at
least one row deposited as part or all of one or more previously
deposited rows. Thus, the mounted substrate 10 has been
repositioned with respect to light source 50 and sensor 64 in order
to image the first droplet set. As the scan continues along a row,
sensor 64 images droplets and provides data to the processor for
analysis as to droplet characteristics (for example, any one or
more of whether a droplet is present, its location, or its size).
The results of the analysis may be compared with the expected
characteristic based on the target array pattern, and used to
identify array errors, stop and/or correct the fabrication process
for subsequent arrays or substrates, or be communicated to a remote
or local user of an array (either as hardcopy printed instruction,
or electronically). Alternatively, the droplets observed can be
part of a test print, and the results used to more carefully set up
the apparatus for depositing actual biopolymer arrays.
Note that in the arrangement of light source 50 and camera 60 in
FIGS. 4 and 5, sensor 64 images a fist droplet set (such as
droplets in a line with position 17a in FIG. 5), whereas light from
source 50 passes through substrate 10 from front side 11a at a
position other than the first droplet set before being reflected by
mirror segment 28 to pass back through the back side 11b of
substrate 10 and the first droplet set. In the situation
illustrated in FIG. 5 the light first passing through from the
first side 11a passes through another droplet set (such as droplets
in a line with position 17b in FIG. 5; sometimes referenced as a
non first droplet set). In this situation, the mirror 27 being
spaced from the back side 11b of substrate 10, allows any shadow
produced by the non first droplet set, to be reduced at sensor 64.
That is, if mirror 27 is at a distance from the substrate 10 that
is far enough outside the depth of field for the line scan camera
60 during observation of an array, any shadows from non first
droplet sets are out of focus and only create a slightly darker
background in the image of a first droplet set. Typically one will
choose the dimensions such that the virtual images generated by the
plane mirror of the drops that one wants to be rejected are two,
three or more times the depth of field away from the nominal plane
of focus. The "depth of field" in this context may be that distance
about the nominal plane of focus in which there is not more than
one pixel size of blur in the case where the sensor has a pixel
size, or in a lens system where the blur is ".epsilon." in the
nominal plane of focus the depth of field may be that distance over
which the blur does not increase to a value higher than .epsilon.2.
In an alternative definition, the depth of field for a lens of
diameter "a" with a focal length "f" and a distance "s" to an
object in the nominal focal plane, is that distance between (s+Dr)
and (s-Df) where:
where where "c" is known as the "circle of confusion" and for the
human eye is typically 0.033 mm.
It will be appreciated that to obtain this effect, mirror 27 need
only be spaced sufficiently from front side 11 by a distance which
depends upon the depth of field of camera 60 (for example, the
spacing might be at least 10 mm, or at least 20 mm, and preferably
at least 30 mm). As referenced before, since mirror 27 is the
actual reflecting surface it does not matter what transparent
material is between that reflecting surface and the array.
In an alternate arrangement illustrated in FIG. 7, light source 50
and sensor 62 may be positioned such that light passes through the
first droplet set when passing through the mounted substrate 10
from first side 11a, and is reflected by mirror 27 to again pass
through the first droplet set. This can be accomplished by using a
beam splitter 150 as illustrated in FIG. 7. In the illustrated
arrangement then, both light from source 50 along axis 130, and the
reflected light along axis 140 will be normal to front surface 11a.
In the arrangement of FIG. 7, when the focused light from source 50
hits the wet droplets, some of the light will be reflected away
from camera 60. The light that passes through the droplets is
reflected off mirror 27 and will be focused again by the lensing
effect of the wet droplets back to camera 60. This results in the
wet droplet appearing as a dark ring with a bright center in the
camera image which allows for excellent droplet imaging in the
inspection process. Although, there is light loss with using beam
splitter 150, the contrast of the droplet images that are captured
by the camera is still very good for proper inspection. Note also
with spacing between mirror 27 and the array as described in
connection with the embodiment of FIG. 4 and 5, shadow resulting
from light initially passing through the first set of droplets
before being reflected, can again be reduced at sensor 64.
Heads 40 may be of a type commonly used in an ink jet type of
printer and each carrying hundreds of ejectors to deposit droplets.
However, it will be appreciated that drop deposition devices other
than heads 40 could be used. In the case of heads 40, each ejector
is in the form of an electrical resistor operating as a heating
element under control of the processor (although piezoelectric
elements could be used instead). Each orifice with its associated
ejector and a reservoir chamber, acts as a corresponding pulse jet
with the orifice acting as a nozzle. In this manner, application of
a single electric pulse to an ejector causes a droplet to be
dispensed from a corresponding orifice (or larger droplets could be
deposited by using multiple pulses to deposit a series of smaller
droplets at a given location). Certain elements of head 40 can be
adapted from parts of a commercially available thermal inkjet print
head device available from Hewlett-Packard Co. as part no.
HP51645A. However, other head configurations can be used as
desired.
As is well known in the ink jet print art, the amount of fluid that
is expelled in a single activation event of a pulse jet, can be
controlled by changing one or more of a number of parameters,
including the orifice diameter, the orifice length (thickness of
the orifice member at the orifice), the size of the deposition
chamber, and the size of the heating element, among others. The
amount of fluid that is expelled during a single activation event
is generally in the range about 0.1 to 1000 pL, usually about 0.5
to 500 pL and more usually about 1.0 to 250 pL. A typical velocity
at which the fluid is expelled from the chamber is more than about
1 m/s, usually more than about 10 m/s, and may be as great as about
20 m/s or greater. As will be appreciated, if the orifice is in
motion with respect to the receiving surface at the time an ejector
is activated, the actual site of deposition of the material will
not be the location that is at the moment of activation in a
line-of-sight relation to the orifice, but will be a location that
is predictable for the given distances and velocities.
The sizes of the features 16 can have widths (that is, diameter,
for a round spot) in the range from a minimum of about 10 .mu.m to
a maximum of about 1.0 cm. In embodiments where very small feature
sizes are desired, material can be deposited according to the
invention in small spots whose width is in the range about 1.0
.mu.m to 1.0 mm, usually about 5.0 .mu.m to 500 .mu.m, and more
usually about 10 .mu.m to 200 .mu.m. Feature sizes can be adjusted
as desired, by using one or a desired number of pulses from a pulse
jet to provide the desired final spot size.
The present methods and apparatus may be used to deposit
biopolymers or on surfaces of any of a variety of different
substrates, including both flexible and rigid substrates. Preferred
materials provide physical support for the deposited material and
endure the conditions of the deposition process and of any
subsequent treatment or handling or processing that may be
encountered in the use of the particular array. The array substrate
may take any of a variety of configurations ranging from simple to
complex. Thus, the substrate could have generally planar form, as
for example a slide or plate configuration, such as a rectangular
or square or disc. In many embodiments, the substrate will be
shaped generally as a rectangular solid, having a length in the
range about 4 mm to 1 m, usually about 4 mm to 600 mm, more usually
about 4 mm to 400 mm; a width in the range about 4 mm to 1 m,
usually about 4 mm to 500 mm and more usually about 4 mm to 400 mm;
and a thickness in the range about 0.01 mm to 5.0 mm, usually from
about 0.1 mm to 2 mm and more usually from about 0.2 to 1 mm.
However, larger substrates can be used, particularly when such are
cut after fabrication into smaller size substrates carrying a
smaller total number of arrays 12.
In the present invention, any of a variety of geometries of arrays
on a substrate 10 may be fabricated other than the organized rows
and columns of arrays 12 of FIG. 1. For example, arrays 12 can be
arranged in a series of curvilinear rows across the substrate
surface (for example, a series of concentric circles or
semi-circles of spots), and the like. Similarly, the pattern of
regions 16 may be varied from the organized rows and columns of
spots in FIG. 2 to include, for example, a series of curvilinear
rows across the substrate surface (for example, a series of
concentric circles or semi-circles of spots), and the like. Even
irregular arrangements of the arrays or the regions within them can
be used, at least when some means is provided such that during
their use the locations of regions of particular characteristics
can be determined (for example, a map of the regions is provided to
the end user with the array). The configuration of the arrays and
their features may be selected according to manufacturing,
handling, and use considerations.
The substrates may be fabricated from any of a variety of
materials, although they should be transparent to the wavelength of
the illuminating light from source 50. In certain embodiments, such
as for example where production of binding pair arrays for use in
research and related applications is desired, the materials from
which the substrate may be fabricated should ideally exhibit a low
level of non-specific binding during hybridization events. In the
present invention the substrates should also be substantially
transparent to the wavelength of illumination during droplet
inspection. For flexible substrates, materials of interest include:
nylon, both modified and unmodified, nitrocellulose,, and the like,
where a nylon membrane, as well as derivatives thereof, may be
used. For rigid substrates, specific materials of interest include:
glass; fused silica; plastics (for example,
polytetrafluoroethylene, polystyrene, polycarbonate, and blends
thereof, and the like).
The substrate surface onto which the polynucleotide compositions or
other moieties is deposited may be smooth or substantially planar,
or have irregularities, such as depressions or elevations. The
surface may be modified with one or more different layers of
compounds that serve to modify the properties of the surface in a
desirable manner. Such modification layers, when present, will
generally range in thickness from a monomolecular thickness to
about 1 mm, usually from a monomolecular thickness to about 0.1 mm
and more usually from a monomolecular thickness to about 0.001 mm.
Modification layers of interest include: inorganic and organic
layers such as metals, metal oxides, polymers, small organic
molecules and the like. Polymeric layers of interest include layers
of: peptides, proteins, polynucleic acids or mimetics thereof (for
example, peptide nucleic acids and the like); polysaccharides,
phospholipids, polyurethanes, polyesters, polycarbonates,
polyureas, polyamides, polyethyleneamines, polyarylene sulfides,
polysiloxanes, polyimides, polyacetates, and the like, where the
polymers may be hetero- or homopolymeric, and may or may not have
separate functional moieties attached thereto (for example,
conjugated).
Arrays produced by apparatus and methods of the present invention
may be used by exposing them to a sample fluid to be tested, in a
known manner, and interrogating the array following such
interrogation. Interrogation can be by any means, such as by
evaluating binding to the features by monitoring fluorescence of
fluorescently labeled targets from them. Of course other means of
interrogation can be used (for example, target bound to features
may be labeled with chemiluminescent or other label). The exposure
to the sample and interrogation may, for example, be at a first
location (such as a laboratory), and the results or a conclusion
based on the results (such as a diagnosis or interpretation of the
results), may be communicated to a location remote from the first
location (such as a doctor's office or patient's residence).
Various modifications to the embodiments of the particular
embodiments described above are, of course, possible. Accordingly,
the present invention is not limited to the particular embodiments
described in detail above.
* * * * *
References